Cytoblock preparation system and methods of use

ABSTRACT

An apparatus and method that may be used for collecting target cells or tissue and preparing a cell block are disclosed.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This is a continuation of U.S. application Ser. No. 11/688,276, filedMar. 20, 2007, which claims priority under 35 USC Section 119(e) fromU.S. Provisional Patent Application Ser. No. 60/852,798 filed on Oct.19, 2006; U.S. Provisional Patent Application Ser. No. 60/846,036 filedon Sep. 20, 2006; U.S. patent application Ser. No. 60/801,759, filed onMay 20, 2006; and U.S. Provisional Patent Application Ser. No.60/783,881 filed on Mar. 20, 2006. This application is related to U.S.patent application Ser. No. 11/284,501 filed Nov. 22, 2005, and U.S.Provisional Patent Application Ser. No. 60/630,870, filed on Nov. 24,2004. The contents of each of the above applications are incorporatedherein by reference in their entireties.

BACKGROUND

Fine needle aspiration (FNA) is a widely used screening diagnosticprocedure. Cells and tissues collected through FNA are used to makesmears for quick staining and microscopic examination at the bed sideand in most cases the collected cells and tissues are used to make acell block for further studies. The current process of FNA is dividedinto two steps and using two separated systems respectively: The firststep is sample collection and the second step is cell block preparation.

A syringe, combined with a fine needle, is conventionally most widelyused as an aspiration tool in current clinical practice for samplecollection.

A fine needle is briefly divided into three parts, the hub, the shaft,and the bevel. The hub is at one end of the needle and is the part thatattaches to the syringe. The shaft is the long slender stem of theneedle that is beveled at one end to form a point. The hollow bore ofthe needle shaft is known as the lumen. Disposable needles should alwaysbe used when preparing admixtures as they are presterilized andindividually wrapped to maintain sterility. Needle size is designated bylength and gauge. The length of a needle is measured in inches from thejuncture of the hub and the shaft to the tip of the point. Needlelengths range from ⅜ inch to 3½ inches; some special use needles areeven longer. The gauge of a needle, used to designate the size of thelumen, ranges from 27 (the finest) to 13 (the largest).

The fine needles used in conventionally FNA procedures have a small hubwith a small space since the main function of the hub is to connect theshaft of needle to the syringe that provide a space for the storage ofcollected cells and tissues. The diameter of the conventional hub isless than 4 mm. During routine FNA procedure using a conventional needleand syringe, frequently, portion of the collected cells and smallfragments of tissues stay in the small space of hub between the shaft ofthe needle and the nipple of the syringe. For majority of the FNAprocedures, the stayed portion of the collected cells and tissuesappears to be critical for the further studies and efficientlyutilization of this portion of specimen remains as a problem. Thecurrent attempt to address this problem is to wash the syringe and theneedle with a fixative (Formalin or ethanol) to remove all stayedspecimen into the fixative in a container, and then separated thespecimen material from the liquid fixative using centrifugationtechnique. After the centrifugation, supernatant (the fixative) is movedout; a matrix (“HistoGel”, argrose gel or others) is added and mixedwith the cell or tissue pellet to make a gel-sample mixture. The tubularcontainer contains the mixture is put in low temperature (40 C.) forminiatures to make the mixture relatively solidified and then is movedout from the container, wrapped by a tissue paper and put into a tissuecassette for further treatment. Since only a small and finite amount ofmaterial can be obtained by FNA and the current process does notmaximally use this limited amount of material. The limited amount ofmaterial collected through this procedure largely inhibits furtherclassification of the disease, which results in more invasive proceduresfor a more conclusive diagnosis. This not only results in increasedcosts, but significantly delays the diagnosis as well.

The need remains for systems and methods which maximize the use of thislimited material for different (H&E staining, immunocytochemistry andother) studies to permit a more conclusive diagnosis to be made by asingle FNA procedure alone and without the need for more invasiveprocedures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic block diagram of an instrument for the preparationof a cytoblock.

FIG. 2 is a schematic of a kit of components for use with the instrumentshown in FIG. 1.

FIG. 3 is a schematic of a centrifuge tube containing a fixativesolution to which cellular material has been added.

FIG. 4 is a schematic block diagram illustrating the centrifugation ofthe centrifuge tube shown in FIG. 3.

FIG. 5 is a schematic block diagram illustrating removal of supernatantfrom the centrifuged specimen by use of a moisture removal device.

FIG. 6 is a schematic illustrating transfer of the cellular pellet fromthe centrifuge tube to a transfer tube after removal of the supernatant.

FIG. 7 is a schematic block diagram illustrating the transfer of thecellular pellet from the transfer tube into a matrix containercontaining a pre-warmed matrix material using a tamping device.

FIG. 8 is a schematic block diagram illustrating the mixing of thematrix material and the cellular pellet using a mixing probe.

FIG. 9 is a schematic block diagram illustrating the cooling incubationof the matrix material/cell pellet mixture to form a gelled specimen.

FIG. 10 is a schematic illustrating the transfer of the gelled specimenfrom the matrix container to the transfer tube.

FIG. 11A is a schematic illustrating the transfer of the gelled specimenfrom the transfer tube to a chamber within a tissue cassette using thetamping device.

FIG. 11B is a schematic illustrating an alternative embodiment of atissue cassette in which the chamber is removable.

FIG. 12 is a schematic illustrating the placement of the gelled specimenwithin a well within an embedding block.

FIGS. 13A-13C are perspective views illustrating various configurationsof embedding blocks and wells.

FIG. 14 is a schematic illustrating the placement of an embedding trayover the embedding block containing the gelled specimen.

FIG. 15 is a schematic illustrating the placement of the embedding trayon a warming plate.

FIG. 16 is a schematic illustrating the placement of the embedding trayon a cooling plate

FIG. 17 is a schematic of an alternative embodiment of a matrixcontainer in which the matrix container takes the form of a syringe.

FIG. 18 is a schematic view of an embedding tray including a dividinginsert.

FIGS. 19A-19C are schematic views of additional embodiments of thetransfer tube of the present invention.

FIGS. 20-27 schematically illustrates other embodiments of the kit ofFIG. 2 according to example embodiments.

FIG. 28 schematically illustrates a collection system according to anexample embodiment.

FIGS. 29-32 schematically other embodiments of the collection system ofFIG. 28 according to an example embodiment.

FIGS. 33-36 schematically illustrate another embodiment of thecollection system of FIG. 28 according to an example embodiment.

FIGS. 37-40 illustrate embodiments of a filter of the collection systemof FIGS. 33-36 according to an example embodiment.

FIG. 41 schematically illustrates a tray device according to an exampleembodiment.

FIGS. 42-54 schematically illustrate other embodiments of the tube ofthe kit a FIG. 2 according to an example embodiment.

FIGS. 55 -65 schematically illustrates additional embodiments of thetube with associated components according to an example embodiment.

FIG. 66 illustrates a transfer forceps according to an exampleembodiment.

FIGS. 67 schematically illustrate different embodiments of a spongeplate according to an example embodiment.

FIGS. 68-71 schematically illustrate other embodiments of the tube ofFIG. 19A according to an example embodiment.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

FIG. 1 shows a device or instrument 10 for the preparation of acytoblock. The instrument 10 provides the mechanical equipment necessaryto process a fine needle aspiration (FNA) specimen for microscopicexamination in a single piece of laboratory equipment. In theillustrated embodiment, the instrument 10 provides a centrifuge 12,supernatant and moisture removal device 14, temperature incubationchamber 16, mixing device 18, a warming plate 60, and a cooling plate19. In the preferred embodiment the instrument 10 is integrallyconnected and unitary in its overall nature. However, it is alsocontemplated that any combination of two or more of the abovementionedcomponents could be formed together as a unitary structure as isconvenient in the laboratory setting.

The instrument 10 is designed for use with additional materials,including disposable and/or consumable components, required forprocessing of the specimen, which are desirably supplied in the form ofa kit 20, as FIG. 2 shows. In the illustrated embodiment, the kit 20provides a centrifuge tube 22 containing a fixative F, a matrixcontainer 24 containing a matrix material M, a transfer tube 26, atamping device 28, a tissue cassette 30, an embedding tray 32, and aparaffin or other embedding block 34. It is contemplated that a kit 20may be provided that includes any or all of these items and that theitems may be provided in any desired quantity. Although in theillustrated kit 20, the transfer tube 26 and the tamping device 28 areshown separately, it is also contemplated that these two items could beprovided together with the tamping device 28 inserted in the transfertube 26, ready for use in preparing a cytoblock sample.

The instrument 10 and associated kit 20 are particularly well-suited foruse in preparing and processing specimens for immunocytochemistry (ICC)studies on the limited materials collected by FNA, biopsy, endoscopicprocedures, washings, and lavages and therefore will be described inaccordance with such use. It will be readily apparent, however, that theinstrument 10 and kit 20 are also suitable for use in other studies,e.g., special stains, in situ hybridization, RNA and DNA studies, aswell as in basic research requiring the collection and saving of treatedcells.

Together, the instrument 10 and kit 20 provide a system that permits thepreparation of multiple sections from a single FNA. Each sectioncontains sufficient cells or material for staining or other studies.

In use, as shown in FIG. 3, the cellular material C obtained from theFNA or other specimen is added to the centrifuge tube 22 containing thefixative F, e.g., formalin. Desirably, the tube 22 has a tapered region36 and a non-tapered, reduced diameter bottom region or chamber 38. Thisconfiguration allows for maximal concentration of cells or specimen atthe bottom of the tube 22 after centrifugation. As will be explainedlater, together with transfer tube 26 and tamp 28, this configurationpermits easy removal and transfer of essentially all of the cellularmaterial.

It should be noted that it is also contemplated that the cellularmaterial C could be added to a centrifuge tube 22 which does not alreadycontain a predetermined quantity of fixative. In such an embodiment, anappropriate quantity of fixative would be added to the centrifuge tube22 along with the cellular material C.

The tube 22 is then placed into the centrifuge 12 for separation. In arepresentative embodiment, the centrifuge 12 is a conventional low speedcentrifuge 12 that permits the separation of the aspirate/fixativemixture into a supernatant S and a cell pellet P, as seen in FIG. 4.

The supernatant S is then removed, as shown in FIG. 5, using supernatantand moisture removal device 14, e.g., with aspiration tubing 40 or otheraspiration means, such that tube 22 retains the pellet P. It isimportant to remove as much of the supernatant S as possible withoutdisturbing the pellet P. In the preferred embodiment the supernatant andmoisture removal device 14 comprises a vacuum device, however alternatemethods of removing the supernatant S are contemplated, including, butnot limited to, using laser or heat provided by the instrument 10. Theinstrument 10 can also include a detector to sense the amount ofmoisture in the tube 22.

The matrix container 24 (which contains a viscous matrix mixture M isdesirably pre-heated by placing the container 24 in the temperatureincubation chamber 16. A variety of matrixes are available in the art,which include agar, agarose gel or “histogel” solid at ambienttemperature, Methocell®, Matrix Gel®, OCT compounds, paraffin, denaturedand non-denatured collagen, fibronectin, laminin, and mixtures thereof.Those skilled in the art will know of other suitable matrixes for cellimmobilization, or will be able to ascertain such, without undueexperimentation. The incubation chamber 16 may be a single chamberselectively adjustable over a broad range of temperature, e.g., between−50-100° C. Alternatively, the chamber 16 may include distinct heatingand cooling chambers (e.g., a separate heating block and cold plate)that are independently adjustable within a defined temperature range,e.g., 50-100° C. and 2-8° C. respectively.

The matrix material M is pre-heated by selecting a temperature thatpermits liquefaction of the matrix. The incubation chamber 16 includes awell (not shown) or otherwise receives the matrix container 24 to heatthe matrix material M to the desired temperature prior to adding thematrix material M to the cell pellet P. It will be readily apparent thatthe chamber may include a series of wells, which may be of the same orof different size and/or configuration, to accommodate multiplespecimens and/or containers 24 of varying size or shape. In oneembodiment, the temperature of the chamber 16 is first set to between90-100° C. to liquefy the matrix material M. Once the matrix material Mhas been liquefied, the temperature is adjusted and lowered to 50° C. tomaintain the matrix material M in the liquid state.

The pellet P is then removed from the centrifuge tube 22 by use oftransfer tube 26 and tamp 28. The transfer tube 26 is a tube having ahollow core 42 and open end 44. The transfer tube 26 is placed into thecentrifuge tube 22 and passed over the pellet P to retain the pelletwithin the hollow core 42, as shown in FIG. 6. The transfer tube 26preferably has a complementary size and shape to the chamber 38 andthereby permits the collection and transfer of essentially the entirepellet P. The tamping instrument 28 desirably has end portion 46 and issized and configured for passage through the transfer tube 26 to releasethe pellet P into the matrix container 24, as shown in FIG. 7. Althoughthe tamp 28 could be formed as a solid piece, the preferred embodimentof the tamp 28 includes a hollow channel along the length of the tamp28, passing through the center of the tamp 28. The hollow channelextends through the end portion 46. This hollow channel allows air to bereleased through the tamp 28. In this arrangement, the matrix container24 desirably contains a pre-measured amount of matrix material toprovide a desired ratio of matrix material M to the cellular pellet P,e.g., a 1:1 ratio.

The tube 26 and tamp 28 may be reusable, e.g., formed of metal, or maybe suitable for disposal after a single use, e.g., formed of plastic.Additional embodiments of the transfer tube 26 and tamp 28 are shown inFIGS. 19 a-19 c. FIG. 19 a shows a transfer tube 126 and tamp 128 whichis similar to the preferred embodiment, however, the tamp 128 has anarrow central portion. The tamp and transfer tube operate in the samemanner as the preferred embodiment, wherein the tamp is pushed toadvance the tamp inside the transfer tube and the tamp is pulled toretract the tamp from the tube.

Tube 226 of FIG. 19 b is similar to tube 126 of FIG. 19 a, however thetransfer tube 226 and tamp 228 include a mating screw mechanism suchthat the tamp 228 is advanced in the transfer tube 226 by rotating thetamp 228 in one direction and retracted from the transfer tube 226 byrotating the tamp 228 in the opposite direction.

Tube 326 of FIG. 19 c is similar to tube 26, however a spring mechanismis engaged between the transfer tube 326 and the tamp 328. In thisembodiment, the tamp 328 is advanced by pushing on the tamp 328 toengage the spring. The tamp 328 is retracted from the tube 326 by againpushing on the tamp 328 to disengage the spring. This mechanism issimilar to that utilized in a spring-retractable ball point pen. Whilethe preferred embodiment discloses utilizing the transfer tube 26, 126,226, 326 and tamp 28, 128, 228, 328 to transfer the cell specimen, it isalso contemplated that the tube 26, 126, 226, 326 and tamp 28, 128, 228,328 could have additional uses in the medical field, such as for takingdermatological biopsies.

The pellet P is then thoroughly re-suspended and mixed within the matrixmaterial M using the mixing device 18. In the illustrated embodiment,the mixing device 18 provides a mixing probe 48, which can be positionedwithin and near the bottom of the tube 24 and activated to providemechanical stirring or mixing motion. It will be readily apparent that avariety of other mixing means may be provided, e.g., a vortex.

With reference now to FIG. 9, the tube 24 is then placed in theincubation chamber 16 for a time period sufficient to solidify thespecimen into a gel G. The chamber 16 receives the tube 24 to cool thematrix material M/cell pellet P mixture to the desired temperature. Thetemperature is desirably selectively adjustable within a range thatpermits solidification or gelling of the histogel, preferably from −2 to−8° C., and more preferably about −4° C.

The matrix tube 24 desirably provides a tapered region 50 and reduceddiameter chamber 52 similar to centrifuge tube 22. Chamber 52 serves toform and maintain the gelled specimen G in a desired shape orconfiguration. In a preferred embodiment, the chamber 52 is of a roundor cylindrical configuration and results in the formation of anessentially round or circular gelled specimen G. It is to be understoodthat the chamber 52 may be variously configured to provide a gelledspecimen G of a desired size and shape, e.g., square or oval.

After solidification, as shown in FIG. 10, the gelled specimen G isremoved from the tube 24 using the transfer tube 26 and tamp 28 or otherremoval means. The transfer tube 26 is placed into the tube 24 andpassed through the specimen G. The tube 26 retains the specimen G withinthe hollow core 42, much like a straw that has been passed through solidgelatin. The transfer tube 26 preferably has a complementary size andshape to the chamber 52 and thereby permits the collection and transferof essentially the entire gelled specimen G and serves to retain thespecimen G in the desired configuration. The tamping instrument 28 canthen be passed through the core 42 to release the gelled specimen G,which can then be further processed, e.g., by frozen section or byembedding in a paraffin or other embedding block. It should be notedthat while paraffin is the preferred embedding material, and is usedthroughout the description of this process, one of skill in the art willrecognize that any suitable embedding material may be utilized.Embedding materials include, but are not limited to nitrocellulose,glue, denatured or non denatured collagen, fibronectin, laminin, gumsyrup, OTC compounds, and various formulations of plastic polymers.

In processing the specimen G by embedding, the specimen G is then placedinto the tissue cassette 30. The cassette 30 may be formed of plastic orother any other suitable material, and may be adapted for multiple orsingle use. As shown in FIG. 11A, the cassette 30 desirably includes arecessed well or chamber 54 to receive the specimen G. Additionalchambers 54 may be provided for additional specimens or quality controlsamples as desired. In an alternative embodiment, illustrated in FIG.11B, a removable basket 56 providing one or more chambers 54 isprovided. The chamber 54 extend through the removable basket 56, formingaperture in the basket 56. A lid or other cover (not shown) may beprovided to cover the chamber 54 and further secure the specimen Gwithin the cassette 30. The chamber 54 is preferably similar andcomplementary in size and configuration to the chamber 38 and specimen Gso as to retain the specimen G in the desired configuration duringsubsequent processing.

The removable basket 56 preferably has at least one cylindrical chamber54 integrally formed therein. However, it will also be readily apparentthat the size, number, and configuration of the chambers 54 of thebasket 56 may be varied to accommodate the procedures being performedand the number and types of specimens being processed. The removablebasket 56 could be made of any suitable material, including, but notlimited to plastic or foam.

After processing, the specimen G is transferred from the cassette 30into a pre-bored hole or well 58 within the embedding block 34, as FIG.12 shows. Block 34 is desirably provided with at least one pre-boredhole to receive the prepared gelled specimen G. FIGS. 13A-13C provide,by way of example and not limitation, possible configurations ofembedding blocks 34 and wells 58. It will be readily apparent that theblock 34 may be of any suitable size and configuration, e.g.,rectangular (FIGS. 13A and 13B) or square (FIG. 13C). It will also bereadily apparent that the size, number, and configuration of wells 58may be varied to accommodate the procedures being performed and thenumber and types of specimens being processed, e.g., a single block 34may provide wells 58 of different sizes (FIG. 13A).

In an alternative embodiment, block 34 may be provided in kit 20 withoutpre-bored wells 58. In this arrangement, transfer tube 26 is preferablyformed of metal or otherwise adapted to bore through the block 34 toform a well 58 or series of wells 58 so that the number and placement ofwells 58 may be determined by user.

The embedding tray 32, which is desirably complementary in size andshape to block 34, is placed over the block (FIG. 14). In theillustrated embodiment, the tray 32 is a conventional embedding tray andmay be formed of metal, plastic or other suitable material and may besuitable for single or multiple use.

The tray 32, containing block 34 with specimen G, is then inverted andplaced on the warming plate 60 (FIG. 15) or otherwise warmed to providesufficient liquefaction to fill and essentially eliminate well 58 andthereby embed the specimen G. The temperature of the warming plate 60 isdesirably selectively adjustable within a range that permits sufficientliquefaction, e.g., from 55 to 65° C.

In an alternative embodiment, wells 58 may be closed and the specimen Gfirmly embedded by pipetting or otherwise delivering heated, liquefiedparaffin without use of the tray 32 (not shown). The paraffin may bepre-heated and liquefied by placing on warming plate 60, microwaving, orother suitable means.

In an additional alternative embodiment, the tray 32 may be providedwith a partitioned insert 62 which includes multiple divisions 64, asshown in FIG. 18. In use, the insert 62 is first placed in the embeddingtray 32. At least one specimen G may then be placed in each partition 64of the insert 62. The specimen G is embedded by pipetting or otherwisedelivering heated liquefied paraffin to the tray 32. The tray 32 is thencooled to form a solidified block 34. Alternatively, the tray 32 can befilled with paraffin before the insert 62 is placed in the tray 32.After the insert 62 is placed in the paraffin on the tray 32, at leastone specimen G can be placed in each partition 64 of the insert. Thetray 32 is then cooled to form a solidified block 34.

The tray insert 62 may have any suitable number and configuration ofdivisions 64. The tray insert 62 may be made of any suitable material,including, but not limited to plastic or metal. This configuration wouldbe particularly useful in creating a cell array containing cell samplesfrom multiple origins, as is further described below.

The block 34 can then be placed on the cooling plate 19 or otherwisecooled to solidify the block 34 (FIG. 16). The temperature of thecooling plate 19 is desirably selectively adjustable within a range thatpermits solidification, e.g., from −50 to +4° C. The block 34 may thenbe and removed from tray 32 for further cytological or histologicalprocessing, e.g., cutting of the prepared block 34 and preparation ofslides for staining or other diagnostic techniques. The cooperating andcomplementary components of the described system, in particular thechambers 38 and 52, transfer tube 26 and tamp 28, and well 58 serve toretain the embedded specimen G in the desired shape, e.g., cylindrical.Because the desired shape has been maintained throughout processing ofthe specimen, a consistent and uniform number of cells or material isprovided on each slide. As a result, more diagnostic procedures may beperformed from a single FNA or other specimen, reducing the need toobtain additional specimens and thereby also reducing the need for moreinvasive procedures. However, it can be appreciated that although acylindrical shape is preferred, the configuration can be in any suitableshape, provided that the chambers 38 and 52, transfer tube 26 and tamp28 and well 58 are all formed with the same desired shape.

FIG. 17 illustrates an alternative embodiment of a matrix container 24Ain which the container 24A takes the form of a syringe. Matrix container24A may be placed in the heating device 16 or otherwise pre-warmed toliquefy the matrix material M. The desired amount of matrix material Mmay then be delivered directly into the centrifuge tube 22 containingthe pellet P (see also FIG. 5). In this arrangement, the container 24Amay include sufficient material M for preparing more than one sample anddesigned for reheating and reuse. In one embodiment, the incubationchamber 16 includes a well (not shown) or other means for holding thecontainer 24A during both warming and delivery of the material M. Thepellet P is then thoroughly mixed and cooled within the centrifuge tube22 as previously described with reference to FIGS. 8 and 9 respectively.The gelled specimen G may then be transferred to the cassette 30 usingthe transfer tube 26 and tamp 28, as also previously described withreference to FIG. 10. It is further contemplated that the matrixmaterial could have a dye added, so that the paraffin is readilydistinguishable from the cell mixture.

While the preferred embodiment of the invention utilizes cells obtainedby fine needle aspiration, it should be clear to one of skill in the artthat cellular material captured by other means could also be utilized tocreate a cytoblock. Cell material could also be collected by endoscopy,including but not limited to arthroscopy, bronchoscopy, colonoscopy,colposcopy, cystoscopy, ERCP (endoscopic retrogradecholangio-pancreatograthy), EGD (esophogealgastroduodensoscopy),endoscopic biopsy, gastroscopy, laparoscopy, laryngoscopy, proctoscopyand thoracoscopy. Cells could also be obtained from lavage procedures,including but not limited to bronchoalveolar, breast ductal, nasal,pleural, peritoneal, gastrointestinal, arthroscopic, and urinary bladderlavages. It is also contemplated that cells could be collected fromcatheters such as those used in infusion, cardiovascular, rental,bladder, urethral, hemodynamic monitoring, neurological, and otherprocedures which would be obvious to one of skill in the art.

It is difficult to screen the expression level of a gene or molecule indifferent cell lines, especially for newly described ones. The currentroutine methods for this purpose include western blot,immunocytochemical study using fluorescence-labeled antibodies,real-time RT-PCR, northern blot, in-situ hybridization, etc.

The current sources of cells for research include commercial orprivately-maintained sources of viable cells in culture, frozen viablecells of specific cell lines, and primary cultured cells derived fromdifferent organs/tissues from different organisms, plants, animalsand/or human. It is very difficult and expensive to maintain these cellsfor scientists and researchers. A “Fixed or Permanent Cell Bank” may beprovided to improve the current system and forms of cell sources. Inthis system, all cells from different possible sources (commercialcompanies, primary cultured cells derived from animals or other sources,etc.) are cultured, collected, fixed with a fixative (formalin, alcohol,et. al) and embedded in paraffin or other materials to form along-lasting (permanent) form of cell source. Based on this principle,different cells can embedded individually like an individual account,and different cell cultures can be together to form a “Cell Bank”.

It is contemplated that a variety of cell lines can be collected andembedded in paraffin blocks 34. Cultured cells may first be embedded inparaffin by conventional or by the above-described methods.

A portion of the paraffin embedded cells may then be taken out byvarious methods (e.g., by use of transfer tube 26 and tamp 28) andre-embedded in a paraffin block 34 as above-described to generateparaffin-embedded cell blocks 34 in a fashion of tissue microarray. Itis preferred that the method of embedding including the embedding trayand partitioned tray insert is utilized to create the cell array.

Various types of arrays could be created by the above described method.By way of example, and not limitation, these types of arrays includeembryonic cell array, adult cell array, primary cell array, cell linearray, tissue array, mammalian array, zoo array, personal cell array,genetically altered array, chemically treated array, or disease cellarray. Further it is contemplated to create a cell array by the abovedescribed method wherein the different cell mixtures differ in one ormore of the characteristics selected from the group consisting ofgenotypic characteristics, species, origin, developmental stage,developmental origin, tissue origin, chemical treatment, cell-cyclepoint and disease state.

The blocks 34 may contain different combinations of different cells fromdifferent systems and organs. By way of example and not limitation,different breast cancer cell lines can be provided in one block,different carcinoma cell lines in one block, different sarcoma celllines in one block, different benign cell lines in one block, differentepithelial cell lines in one block, and different mesenchymal cell linesin one block. It is also contemplated to create a cell array with cellpopulations from several different types of body tissues in one cellarray, the tissues including but not limited to blood, muscle, nerve,brain, heart, lung, liver, pancreas, spleen, thymus, esophagus, stomach,intestine, kidney, testes, ovary, hair, skin, bone, breast, uterus,bladder, spinal cord, and body fluids.

Cells from many cell lines, including cells from primary cultures, cellsfrom humans, rats, mice, and other animals, cells from differentorganisms, and cells from an organism at different stages ofdevelopment, may thereby be provided in a single cell block. The cellsmay be treated with different conditions (different chemicals, differenttemperatures, different culture conditions, etc.) based on specificrequirements, collected, and embedded in a single block 34.

A variety of cell lines may be maintained as a “cell bank” and blocks 34containing specific cell lines may be pre-formed and provided as “readyto use” blocks 34 to researchers or others. Pre-made blocks 34 includingthe desired embedded specimens or cell lines may be customized (e.g.,specific cell line(s) and number of wells) and manufactured according tothe user's specific needs.

Sections can then be generated from different blocks 34 and slidescontaining the cells from these sections can be obtained and processedas desired, e.g., protein, DNA, RNA, or other studies.

FIGS. 20-27 illustrate other embodiments of centrifuge tube 22. FIG. 20illustrate tube 422. Tube 422 is similar to 22 except that tube 422 hasan open end 423. As a result, the removal of supernatant and fluid fromtube 422 is facilitated using gravity.

FIG. 21 illustrates filtering unit 522. Filtering unit 522 is similar totube 422 except that filtering unit 522 additionally includes filter 523positioned within opening 423 of tube 422. Filter 523 may comprise apiece of specific glass wool, glass fiber, paper, membrane, plastic ormetal net with or without an additional supporting web. This filter willhave a certain cut off spaces so that it can hold the cells, celldebris, cell organelle or cell secretion materials but will allow thewater run through the filter at certain speed of centrifugation.However, the filter can be designed to hold the mix of cell and matrixat the regular condition (without centrifugation force) or supplied by avacuum system at the bottom or using a tamp to push the fluid runningthrough the filter. The filter can be fixed on the bottom of the tube orremovable from the open-ended tube.

In other embodiments, filter 523 may be provided with a cut off spacesor filter openings having a size and density so as to allow the fluid topass through, even without centrifugation. After the fluid runningthrough its the filter, the filter 523 will be covered and sealed by alayer of material (gel, Vaseline, plastic tape, paper tape, et. al.) orthe spaces of the filter will be filled by specific materials (gel,Vaseline, petrolatum, et. al) from the bottom of the filter and in thisway the matrix and the mixture of cell and matrix will be hold insidethe tube without leakage.

FIG. 22 illustrates filtering unit 622. Filtering unit 622 is similar tofiltering unit 522 except that filtering unit 622 additionally includesa plug 629. Plug 629 is coupled to filter 523 and secures filter 523 atthe bottom to the open end of the tube 422. Plug 629 is coupled to tube422. In particular embodiments, plug 629 is coupled to tube 422 indifferent manners such as with a friction fit, snaps, hooks, screwthreads in the like. For example, FIG. 23 illustrates filtering unit 722in which plug 629 is coupled to tube 422 by a friction fit (pushing).FIG. 24 illustrates filtering unit 822 in which plug 629 is coupled totube 422 via screw threads.

In operation, as shown by FIGS. 25 and 26, after the fluid has passedthrough the filter 523 (as shown in FIG. 28), a sealing material 625 maybe placed across filter 523. Sealing material 625 may comprise a tape,gel material or other materials which can prevent water leakage can beused to seal the any open spaces in the filter.

Alternatively, as shown by FIG. 27, a second plug 628 which containsmaterials 627 such as petroleum jelly (Vaseline) or sticky tape orothers can be used to cover the first plug from the outside. Then thematrix material can be added in the tube and a cell-matrix mixture willbe made. After cooling down at lower temperature (4° C.), the mixturecan be moved out by either punching out or by a different way, forexample, move the plugs out of the tube to let the end of tube openagain and pushing the mixture out from the open end by using a plungerfrom the top to the bottom of the tube 422. The mixture will be directlyplaced in the hole of the specifically designed cassette.

FIG. 28 illustrates the collection of supernatant and fluid after it haspassed through filter 523. In particular, FIG. 28 illustrates tube 422removably coupled to a collection system 927. Collection system of 927is configured to collect fluids after they have passed through filter523 such that the collected fluids may be reused in case there is aleakage of the filter or for other purposes. In the embodiment shown inFIG. 28, collection system 927 includes container 929. As shown by FIG.28, container 929 has a mouth 931 configured to receive a lower end oftube 422. In the example illustrated, mouth 931 is configured to supporta shoulder 933 of tube 422 during filtering. In other embodiments, thesize and shape of the container 929 can be different. Container 929 canbe made of different materials.

FIGS. 29-32 illustrate other embodiments of collection system 927. FIGS.29-31 illustrate collection system 1029. As shown by FIGS. 29 and 30,collection system 1029 includes vacuum device 1031 and plug 1042, vacuumdevice 1031 comprises a bottle-like device with a narrower upper openend. The shape of the opening can be round or other specific shapes. Thelower portion of the device is larger and the bottom end is also opened.It also could be in different shapes and sizes. But the edges of theopenings should be smooth. At one side of the device, there is a hole oropening which can be used to connect to the vacuum 1041.

As shown in FIG. 30, plug 1042 is designed to cover the upper opening ofthe bottle-like device. The plug 1042 is divided into two portions. Thelower portion has an exact size and shape to fit into the upper openingof the device 1031 without air leakage from the connection areas. Theupper portion of the plug 1042 is designed with a specific size andshape to fit the tube 422. There is a central opening 1043 in the plug1042 to allow portion of the tubes fit in and allow the fluid runthrough at a vacuum. There could be a tube 1044 with one end locatedinside the lower portion of the central hole of the plug, with anotherend toward to the bottom of the device. The length of the tube can bedifferent.

FIG. 31 illustrate collection system 129 assembled. As shown by FIG. 31,collection system 1029 additionally includes support 1033 collectioncontainer 1030. In the example embodiment illustrated, support 1033comprises a flat, soft and water-proof cushion (or mat or pad). The sizeof the cushion should be larger than the lower end of the bottle-likedevice.

Collection container 1030 collects fluid drawn through plug 1042 as aresult of vacuum applied to an interior of device 1031. In practice,container 1030 is positioned at the center on the cushion, and put thelower end of the device on the cushion with the central tube toward thecontainer. When the lower end of the bottle-like device is put on thiscushion, the bottom of the device is automatically sealed and there isno air leakage from the connation between the device and the cushion. Inthis case, a vacuum will be generated inside the device if the vacuum issupplied through the side opening of the device. The bottle-like device1029, plug 1042 and the cushion 1033 can be made of different materials.They could be made of glass, plastic, rubber, fibers, and othermaterials.

FIG. 32 illustrates collection system 1129, another embodiment ofcollection system 1029. Collection system 1129 is similar to collectionsystem 1029 except that collection system 1129 includes container 1131in lieu of container 1031. Container one 131 is similar to container1031 except that container 1131 has a closed bottom so the fluid fromeach sample cannot be recollected individually. Alternatively, as shownby FIG. 32, a port or opening 1133 can be made at the lower portion ofthe device and a tube 1135 can be connected to the opening to allow thecollected fluid run out of the device.

FIGS. 33-36 illustrate filtering unit 1222 and collection system 1229.Filtering unit 1222 and collection system call 29 are configured totreat larger volumes of specimens. Filtering unit 1222 comprises anotherembodiment of filtering unit 522. As shown by FIGS. 33-35, filteringunit 1222 comprises a double-tray device that is composed of two trays1240, 1242 with either identical or similar shape so that one tray canbe closely attached to another one. But the two trays could be indifferent size from each other. The central and also the bottom areas ofthe trays are opened. There is a filter 1223 (A piece of specific paper,membrane, plastic or metal net or other materials) is placed between thetwo trays and cover the opened areas of the trays. This tray-filter-traysandwich type device can be used as a unit. The upper portion of theplug of the vacuum system can be designed to fit the outside tray sowhen the double-tray device is put on the plug, they fit each other verywell without air leakage.

As shown by FIG. 36, collection system 1229 is similar to collectionsystem 1029 except that collection system 1229 includes plug 1250, whichis similar to plug 1042 except that plug 1250 is specifically configuredto at least partially received and support filtering unit 1222. Asfurther shown by FIG. 36, once the filtering unit 1222 is put on theplug 1250 and the vacuum system begins to work, the sample will be addedto the tray and the fluid with run through the filter and the tissue orcell materials will be hold by the filter. The inner tray will be movedout, the materials remained on the filter will be wrapped up using thesame filter and will be put into the cassette for next treatment. Thetrays can be in different size and shape and made of different materials(plastic, paper, filter paper, fibers, and others).

Alternatively, a single tray device can be made with one of thedescribed trays covered with a filter; however, there is no second trayto cover the filter. In both devices, the filter is removable from thetray and can be wrapped.

FIGS. 37-40 illustrate other embodiments of filter 1223. In particular,FIGS. 37-40 illustrate filter 1323. Filter 1323 is pivotable areactuatable between an open position and a closed position thus to form aclosed or sealed volume. The filter material (clothe, fiber, plastic,metal et. al) is attached with a strip of fastener. The filter can befolded and sealed by the fastener 1251 to form a bag-like structurewhich can be opened and unfolded in need. The sealed, bag-like filtercan be put into the cassette directly. Alternatively, the filter can befolded and sealed by a sewing machine-like device, a stapler, a heaterdevice or other techniques. In the example illustrated, perimeterportions of filter 1323 include opposite portions of a hook and loopfastener system (VELCRO). In other embodiments, other permanent ornon-permanent attachment or fastening devices may be employed in lieu ofVELCRO.

FIG. 41 illustrates an embodiment in which a single tray device 1322 canbe made in a fashion of funnel: the bottom potion of the tray is openedwith a protruded edge 1350 in a specific size and shape that allows theedge be put into the tissue cassettes 1351 directly without filter. Inthis case, the fluid which contains the small tissue fragments can bepoured directly into the funnel-like single tray and the fluid will runthrough the bottom of the cassette and the tissue fragments will becollected directly inside the cassette.

These devices can not only used to treat cytology specimens, but alsocan be used to treat specimens of surgical pathology such asendocervical curettage and other surgical curettage specimens.

Alternatively, instead of using the vacuum system, the tubes and trayscan be directly put on papers, paper towels, sponges or other liquidabsorption materials to help the fluid run through the filters.

FIG. 42 illustrates modified sample tube 1422. Tube 1422 is similar tofiltering unit 622 except that the plug 1424 contains a hole (ormultiple small holes) 1425 and a protruding area 1426 which may functionas a part of screw and as a holder and it can be used to separate theplug from the tube by pulling the plug out of the tube. The lumen of thetube has a non-tapered, reduced diameter bottom region or chamber,called sample collecting chamber 1427.

FIG. 43 illustrates modified sample tube 1522. Tube 1522 is similar tosample tube 1422, the centrifuge tube 22 and filtering unit 622 exceptthat 1422 contains only the non-tapered, reduced diameter bottom regionor chamber 1538 with a non-tapered, reduced diameter, cylindrical lumen,called as sample collecting chamber 1527. This chamber has been descriptand labeled as 38 in the tube 22 in FIG. 3.

FIG. 44 illustrates a frame of device 1641 which contains a barrel 1642,a inner needle 1643, a valve 1644 with a hole 1645, a sample tube holder1646 with a screw 1647 and a septum 1648 with a hole 1649.

Sample tube 1422 can be directly connected to a frame of the device 1641in a manner of screw system composed of the protruding area 1426 andscrew 1647 in the frame. The hole 1425 in the plug 1424 will connect tothe hole 1649 in the septum 1648 of the frame 1641. The vacuum tube 1650is then put inside the barrel 1642 of the frame. The inner needle 1643will penetrate the cover 1651 of the vacuum tube. When the valve 1644 ofthe frame is located at a “open” position, the hole 1645 will beconnected to the hole 1649 in the septum 1648 and the vacuum will betransferred to the lumen 1427 of the sample tube 1422. As a result, thefluid will run through the filter (membrane) and be collected in thevacuum tube. At the same time the cell, cell debris, cell organelleand/or tissue fragments will be held by the filter (membrane). In thisway, the cell and/or tissue materials will be separated by aready-to-use vacuum system. After this separation, sample tube 1422 willbe separated from the frame and the hole 1425 will be sealed by asealing material. Sealing material may comprise a tape, gel material orother materials which can prevent water leakage can be used to seal theany open spaces in the filter. Alternatively, a second plug whichcontains sealing materials such as petroleum jelly (Vaseline) or stickytape or others can be used to cover the first plug from the outside.Then the matrix material can be added in the tube and a cell-matrixmixture will be made. After cooling down at lower temperature (4° C.),the mixture can be moved out by either punching out or by a differentway, for example, move the plugs out of the tube to let the end of thetube open and pushing the mixture out from the open end by using aplunger from the top to the bottom of the tube. The mixture will bedirectly placed in the hole of the specifically designed cassette. Thecollected fluid in the vacuum tube can be used for different testsincluding pH, chemical, biochemical, immunological, molecular and otherstudies. Though a vacuum control valve is preferred in this device tocontrol the vacuum achieving and releasing, the device can be designedwithout the vacuum control valve in order to cut the cost of themanufacture.

In a different setting, sample tube 1522 contains only the non-tapered,reduced diameter bottom region or chamber 1538 with a non-tapered,reduced diameter, cylindrical lumen-sample collecting chamber 1527.Sample tube 1522 can be put inside a tube holder 1660 in a manner thatthe top of the tube 1528 faces the bottom wall 1661 of the tube holder1660. The sample collecting chamber 1527 will connect the central lumen1665 of the nipple 1664 of the tube holder 1660. Then the frame of thedevice 1641 can be tightly connected to the tube holder 1660 byscrewing. Then the vacuum tube 1650 will be put inside the barrel of theframe as described above. A vacuum control valve 1644 will be used tocontrol the vacuum. When the valve is located at a “open” position, thehole 1645 of the valve will connect the hole 1649 of the septum 1648 andthe vacuum will be supplied to the sample collecting chamber, thecentral lumen of the nipple and the needle. When the vacuum valve is ata “close” location, the hole 1645 will be disconnected to the hole 1649and the vacuum will not be supplied to the sample collecting chamber andthe needle. When the needle is inside the target tissue during aprocedure of aspiration, the valve can be put in the “open” location andthe vacuum provided from the vacuum tube will transferred to the tissueand cells and tissue fragments will be sucked into the sample collectingchamber of the sample tube B through the needle. The cells, cell debris,tissue fragments will be held by the filter (membrane) of the sampletube. Fluid aspirated from the tissue will pass through the filter andcollected in the vacuum tube. As a result, the target cells and tissuesfragments will be separated from fluid during the procedure ofaspiration and will be ready to make a cell block. Once the procedure isdone, the vacuum control valve will be placed at a “close” position. Thevacuum in the sample tube and the needle will be released. The needlecan then be moved out from the tissue without cell and tissue fragmentsbeing reflux back through the needle to the target tissue. The devicewill be placed in a position with the needle facing up. The sample tubeholder will be separated from the frame of the device and the sampletube 1522 will be moved out from the device frame. The hole 1525 will besealed and a cell-matrix mixture will be made as described above.

While the preferred embodiment of the invention utilizes cells obtainedby fine needle aspiration, it should be clear to one of skill in the artthat cellular material captured by other means could also be utilized toseparate cells and tissue fragments from fluid and create a cytoblock.For example, the nipple 1664 of the tube holder 1660 can be connected toa catheter or other tubes, instead of a needle. In this way, the devicecan be used to collect cells and tissue fragments from other proceduressuch as endoscopy, including but not limited to arthroscopy,bronchoscopy, colonoscopy, colposcopy, cystoscopy, ERCP (endoscopicretrograde cholangio-pancreatograthy), EGD(esophogealgastroduodensoscopy), endoscopic biopsy, gastroscopy,laparoscopy, laryngoscopy, proctoscopy and thoracoscopy. Cells couldalso be obtained from lavage procedures, including but not limited tobronchoalveolar, breast ductal, nasal, pleural, peritoneal,gastrointestinal, arthroscopic, and urinary bladder lavages. It is alsocontemplated that cells could be collected from catheters such as thoseused in infusion, cardiovascular, rental, bladder, urethral, hemodynamicmonitoring, neurological, and other procedures which would be obvious toone of skill in the art.

The vacuum control valve 1644 can be designed in different forms andlocated in different locations of the device. FIGS. 45(A) and 45(B)illustrate two examples of these different valves: Pushing valve 1744and rotating valve 1844. FIG. 54 illustrates a rotating valve comprisingof different components. The vacuum position indication window 1846 isdesigned to demonstrate the “opening” and “closing” of the vacuum byshowing different colors. For example, a green color indicates an “open”of the vacuum and a red color indicates a “close” of the vacuum. In thepresenting drawings, the valve 1644 is located in the septum 1648 and ismore close to the end which connects the sample tubes. Alternatively,the valve can be located at different locations of the frame of device.The valve 1844 is composed of different parts including a vacuumposition indication window 1846, a hole 1847, gear wheel 1848, softplastic cover 1849, spring 1850, levels for spring 1851, spring 1852,hole to inner needle 1853, barrel 1854, step stopper 1855, steps 1856,axis 1856.

A vacuum indicator will be put into the system, either in the vacuumtube or associated with the vacuum valve. This vacuum indicator mayreflect the real vacuum status of the vacuum tube or/and the samplecollecting tubes. This indicator may be designed based on differentmechanisms such as color change, volume change of a ball shapestructure, electronic signal or/and transferred digital signal and otherpossible forms. The vacuum indicator is an important part of the device.

FIG. 46 illustrates the connection of sample tube 1422 to the frame1641. The tube 1422 can be tightly connected to the device frame 1641 bya screw system 1426 and 1647. The plug 1424 can be tightly attached toone side of the septum 1648 of the frame 1641. The hole 1425 in the plug1424 can match and connect to the hole 1649 in the septum 1648 to formone channel.

FIG. 47 illustrates a vacuum tube 1650 which has a rubbery cover 1651and a tube 1652 which contains vacuum.

FIG. 48 illustrates a connection between the vacuum tube 1650 and theframe 1641. The vacuum tube 1650 can be put into the barrel 1642 of theframe 1641. The inner needle 1643 will penetrate the cover 1651 of thevacuum tube. When the valve 1644 is moved to a position to allow itshole 1645 connect to the holes in the septum, the vacuum will betransferred from the tube 1650 through the inner needle 1643, the hole1649 in the septum, the hole 1645 in the valve and the hole 1425 insample tube 1422 or 1525 in the sample tube 1522, the membrane 1423 andfinally to the channel 1427 or 1527 of the sample tubes.

FIG. 49 illustrates a tube holder 1660 which has a bottom wall 1661, aside wall 1662, and a screw 1663 at the outer surface of the side wall,and a nipple 1664. The bottom wall 1661 is round and has a flat surface.The nipple 1664 is located in the center of the bottom wall and has acentral channel 1665. The nipple can be used to connect the needle 1666.The side wall 1663 is round in shape and the outside screw can be usedto connect the frame 1641. The side wall and bottom wall together form achamber which can hold the sample tube B 1522.

FIGS. 55A-C illustrate a collecting tube 2122 (2122 A, B and C). In FIG.55A, tube 2122 A is a modified embodiment of tube 22 and has a taperedregion 2136 and a non-tapered, reduced diameter bottom region or chamber2138 with a flat plane floor 2123. FIG. 55A illustrates the tube 2122 Awith a needle shaft 2104 penetrating through the flat floor 2123 andprotruding into the inner space of the chamber 2138 with a upper portionof the shaft 2114 located above the floor. Briefly, this upper portion2114 of the shaft 2104 has two specific functions: 1) prevent the refluxof collected specimens back through the shaft; 2) make a dot marker onthe bottom surface of a cell-matrix mixture block which can be used asan orientation marker in the embedding process. Basically, the cellmatrix mixture is made and maintained in a cylindrical shape and thecells are located at the area of bottom surface and this bottom surfaceneeds to be embedded on the cut surface of the final paraffin block. Itis easy to confuse the top and bottom surfaces during the processbecause they look similar to each other. With the presence of upperportion 2114, the 2114 will occupies a small space in the bottom portionof the mixture G and a small hole (dot marker) will be generated whenthe mixture G is moved out of the tube and this small hole will beworked as a marker of the bottom surface. The small hole itself can beused as a orientation marker. The bottom surface can also be gentlyinked with a specific ink and this hole will catch the ink and highlightthe bottom surface.

In FIG. 55B, tube 2122B is a different embodiment of tube 2122 A. Tube2122B has a screw 2102 on the outer surface of the upper portion of thetube 2103. The screwing surface will help the tube to connect to aspecifically designed syringe or other vacuum system by the screw.

In FIG. 55C, tube 2122C is a different embodiment of tube 2122A. Tube2122C has a “ear” or “shoulder” protruding portion 2118 on the top ofthe tube. The portion 2118 works as a part of the lure lock to connectto a specifically designed syringe. A supporting tube 2119 can also beadded to the system. This tube has two functions: 1) support the tube2122 A, B, C when the tube is centrifuged to separate the supernatantand the cells; 2) shelter the needle and collect the possibly leakedmaterial from the tube.

FIG. 56 illustrates specifically designed frame of vacuum device 2141.The frame 2141 has a barrel 2142, a piston 2143 and a connecting portion2146 and a screw 2147 on the inner surface of the connecting portion2146. In FIG. 56A, a septa 2148 is located to separate the barrel andthe connecting portion and there is an opening 2149 at the center of thesepta 2148. In a different embodiment, a piece of cushion 2130 can beplaced on the lower surface of the septa and this cushion will directlycontact with a upper edge of the tube 2122 (A, B, C) to better seal theconnecting point between the septa and the upper edge of the tube. FIG.56 B illustrates another embodiment of the vacuum device. The septa isreplaced by a nipple 2131 which combined with the connection portion2146 and a screw 2147 to form a lure lock which then can be connected totube 2122 through the portion 2118. The diameter of the nipple will befit the size of the tube 2122C. In another embodiment, a filter membrane2132 can be put on the opening of the nipple to avoid the collectedspecimens leaking to the barrel of the vacuum device through the opening2149 A of the nipple.

FIG. 57 illustrates different embodiments of tube 22. FIG. 57 Aillustrates tube 2222 with a frame 2422 and an open bottom 2423.

FIG. 57B illustrates a flat plane 2223. The frame 2422 and the flatplane 2223 can be mounted together by different methods to form a tube2222A as illustrated in FIG. 57D.

FIG. 57C illustrates a plug 2225 which is considered as a differentembodiment of plane 2223. Plug 2225 can be mounted to the frame 2422 Bto form a tube 2222 B with a flat bottom surface by pushing the tubeframe into the plug. A cushion 2230 can be placed on the inner surfaceof the plug to seal the connection between the frame and the plug moreefficiently.

FIG. 57F illustrates a different embodiment of the connection betweenthe frame 2422 C and the plug 2226 by a screw type connection.

FIG. 57G illustrates a different embodiment of the flat plane 2223. Inthis embodiment, a needle shaft 2104 penetrates the flat plane 2223 withits upper portion 2014 located above the plane.

FIG. 57H illustrates a different embodiment of the upper portion 2014 ofthe shaft 2104. In this embodiment, the upper portion of the shaft has acurve 2015. The upper opening of the shaft is located above the plane2223 and this location will avoid the re-flux of collected specimenthrough the shaft. The curve 2015 will help to guide the collectedspecimen stored along a side wall of the tube to avoid spreading duringthe aspiration. The needle shaft can be located at different areas ofthe flat plane including a central or an eccentric location or at theedge.

FIG. 58 illustrates different embodiments of the connection between thechamber 38 and the plug 2225. FIGS. A, B, C and D illustrate differentembodiments of chamber wall 2238 A, 2238 B, 2238 C, and 2238 D withmatched plugs 2225A,2225 B, 2225 C and 2225 D, respectively. The chamberwall 2238 can also be formed of two parts with a joint formed across thewhole chamber. As illustrated in FIG. 57A.

FIG. 59 illustrates different embodiments of the tube 2222 with itschamber mounted to a plug which carry a needle.

FIG. 60 illustrates a different embodiment of the system. In thisembodiment, only the chamber portion 38 of the tube 22 will be employed.

FIG. 60A is a copy of the FIG. 42 in the application U.S. 60/846,036.And it was described as the follows:

FIG. 60A illustrates a specimen collector 2002. Collector 2002 isdesigned to contain three parts including a metal shaft 2004 with acentral lumen 2006, a sharp bevel 2008 and a hub 2010, as in a needle.The most important difference between this novel collector and aconventional needle is that the hub of the collector is designed in aspecific size and shape to enable the hub itself function as a collectorof specimen, instead of functioning only as a connector between thesyringe and the shaft of a syringe in a conventional needle.

The example embodiment of the hub is in a cylindrical shape, though itcould also be in other shapes. The bottom 2016 of the hub should be flatand in a round or other shape. The inner space 2012 of the hub should beat least 4 mm in diameter (The hub of a conventional needle has an innerspace with a diameter up to 4 mm). The length of the hub is variable (2to 25 mm) depending on specific requirement of different procedures andtarget organs. The upper portion of the metal shaft 2014 can be locatedin a manner that it penetrates the bottom of the hub 2016 and protrudesinto the inner space 2012 of the hub with a variable length of theprotrusion (0 to 20 mm). The shaft 2004 and its upper portion 2014 canbe located at variable location of the bottom 2016 of the hub includinga central location or an edge, or eccentric location. The upper portion2014 of the shaft can be either straight or with a curve to direct thecollected material to a sidewall of the hub. The hub may has a “ear” ora “shoulder” 2018 which may works as a part of a lure lock to connectthe collector 2002 to a vacuum system such as a syringe. The collectorcan also be connected to a vacuum system by gel, glue or otherchemicals. The collector will be separated from the vacuum system byusing a physical force or other methods, after the FNA procedure.”

FIG. 60B illustrates the curved upper portion 2015 of the needle shaft.The upper portion of the needle may be curved to guide the collectedcells or tissue fragments toward a side of the chamber of the tube. Thiscovered portion will prevent the cells from running up into the barrelof the vacuum device. The curved portion 2015 could be a part of theneedle shaft. Alternatively it could be extended out from one portion ofthe proximal end of the needle shaft as an “umbrella” or “cover”.Alternatively, the “umbrella” or “cover” structure can also be extendedup from the floor adjacent to the proximal opening of the needle.

FIGS. 60C and D illustrate examples of different embodiments of thechamber using the same principle illustrated in FIGS. 57, 58 and 59.FIG. 60D illustrates an embodiment where metal shaft or needle 2004 hasan upper end adjacent to the floor or bottom 2026 and where a cover 2027extends across and over the central axis of lumen 2006. As a result,cells and/or tissue drawn through lumen 2006 is directed in a sidewaysdirection to reduce the potential for tissue loss caused by such cellsor tissues being drawn further up into the barrel of a syringe. In oneembodiment, the cover 2027 is formed as part of the needle 2004. Inanother embodiment, the cover 2027 is formed as part of the floor orbottom 2026 (shown in FIG. 60D).

FIG. 61 illustrates a syringe type vacuum system 2032. The vacuum system2032 contains a barrel 2038, a plunger 2040 and lure lock 2036 and anipple 2034. The diameters of the nipple and the lure lock should bevariable to fit the hub 2002. In addition, a filter membrane 2035 may beplaced at the lower opening of the nipple to avoid the collectedspecimens leaking to the barrel of the vacuum device through the openingof the nipple.

FIG. 62 illustrates a different embodiment of “Modified tube for vacuummethods” as illustrated in FIGS. 20 to 32. In this embodiment, the plug2625 in FIG. 62 B contains a flat bottom 2627 with central opening 2649.A filter membrane 2630 is placed on the inner bottom surface.

FIG. 62C illustrates a tube 2622 A formed by mounting the plug 2625 withthe tube 2622.

FIG. 62D illustrates a different embodiment of vacuum device 1029 asdescribed in FIGS. 29 and 31. In this embodiment, a vacuum device 2629has a barrel 2636, a bottom 2631, with extended stand 2632. On the upperinner surface of the barrel, there is a screw 2633. On a side wall,there is an opening plug 2634 with a “ear” or “shoulder” 2635 which canconnect to a syringe by a lure lock.

FIG. 62E illustrates a connection between the tube 2622A and the vacuumdevice 2629 in a fashion of forming a screw 2636.

FIG. 62F illustrates a different embodiment of the tube 2622. In thisembodiment, the connection between the frame of the tube and the plug isin a screw type.

FIG. 63 illustrates different embodiments of vacuum valves which areillustrated in FIG. 45A and FIG. 45B. In these embodiments, there is noinner needle.

FIG. 64 illustrates a different embodiment of sample tube B 1522illustrated in FIG. 43 and a tube holder 1660 illustrated in FIGS. 49and 50. In this embodiment, the sample tube 1522 A is same as 1522.There is no nipple present in the tube holder 1660 A. Instead, a needleshaft 1604 A penetrates through the flat bottom floor 1661 A and withthe upper portion 1614 A protruding into the inner space 1527 A of thesample tube 1522 A.

FIG. 65 illustrates a portable embodiment of incubation carrier orchamber 16 as illustrated in FIG. 8. In this embodiment, the innerincubation chamber 2316 is battery powered and is portable. It containsa battery 2317, an electric resistance 2319 which is connected to thebattery by a connecting wire 2318, a heating chamber 2320 with an innerspace 2324. A matrix container 2326 containing matrix 2328 can be placedinside the inner space 2324 of the heating chamber 2320. It alsocontains a switch (not show in the picture) to adjust the temperature ofthe chamber. The temperature can be adjusted from 0 to 100 degrees C.

FIG. 66 illustrates a specially designed forceps used to transfer therelatively solid cell-matrix mixture G from the tubes to a specificallydesigned cassette as illustrated in FIGS. 8 to 10. In this embodiment,as illustrated in FIG. 66 A, the forceps 80 has two arms 85 which jointtogether by a joint 84. The lower portion of the forceps 81 hassemi-annular shape. There are spaces between the two arms. The lowerportion 81 of the two arms can form a cylindrical shape by pushing thetwo arms toward each other. The diameter of the cylindrical spacebetween the two arms should match with the diameter of chambers 38,1522, 2138, 2238, 2012, 1527, and 1527 A.

FIG. 66B illustrates a bottom view of the lower portion of the forceps80. The bottom 82 should be flat.

FIG. 66C illustrates a inner face of the lower portion of the arms.There are some teeth-like structures 83 on the inner face to helpholding the mixture G during the transferring process.

FIG. 67 illustrates different embodiment of specifically designedcassette 30 as illustrated in FIG. 11A and FIG. 11B. In this embodiment,cassette 30A has slits or openings through its floor to permit passageof fluids therethrough and further contains a sponge plate 31A with aopen spaced chamber 54A. The chamber 54A should have a shape and sizematched with the mixture G to maintain the shape and orientation of G.In another embodiment, there may be a web or filter paper being placedunder or/and above the sponge plate to cover the sponge chamber in orderto prevent possible loss of cells in the mixture G during the process.In other embodiments, plate 31 may be integrally formed as a singleunitary body with a remainder of cassette 30.

FIG. 67C illustrates a different embodiment of the sponge plate 31Bwhich contains multiple chambers 54 B and an orientation marker 32. Themarker can be a defect of the sponge plate (for example a defect at onecorner), an inked area, or specific labeling.

FIG. 68 illustrates a different embodiment of a transfer tube 26, 126,226, and 326 as illustrated in FIGS. 10, 11A, 19A, 19B and 19C,respectively. In this embodiment, the transfer tube 126A contains a tamp128A with central opened lumen 130A with or without a structure 129A atthe bottom of tamp. A vacuum can be employed through the central lumento the bottom of tamp and the upper surface the mixture G to helpholding the mixture G during the transferring process.

FIG. 69 illustrates another embodiment of the transfer tube of 126. Inthis embodiment, there is a thin layer, flexible narrow plate likestructure 132 is placed along the wall of the tube 126 B with a handle131 which can move along the tube. After punching the mixture G into thetube 128B, the handle 131 will be moved down along the tube wall. Thelower end of the structure 132 will encounter with the bottom surface ofthe chamber 38 and be bended toward the center of the chamber along thebottom surface of the chamber 38. This bended portion 133 of thestructure will help to move the mixture G from the chamber and betransferred to a cassette. The structure 132 can be designed as a singleplate as illustrated in FIG. 69. It can also be multiple and locatedtoward to different directions. It may be located inside or outside thetransfer tube or even inside the wall of the tube.

FIG. 70 illustrates a different embodiment of tube 22. FIG. 70Aillustrates a modified tube 2722 contains a barrel 2701, a taperedregion 2736 and a non-tapered, reduced diameter bottom region or chamber2738, a flat floor 2725. In addition, a needle shaft 2704 penetrates theflat floor 2725 and with or without the upper portion 2714 protrudinginto the inner space 2711 of the chamber 2738. The distance between theupper opening of the shaft and the inner surface of the floor 2725 isvariable (0-1.5 cm) depending on different situations. The upper portion2714 could be either straight or curved as described above. In addition,a piston 2705 is also a part of the embodiment. The piston has a centralcolumn 2706 with a tapered region 2707at the lower portion of the columnand a flat bottom 2708. This piston could be in different shape and madeof different materials, though the embodiment 2705 is preferred. Thepiston is in a size and shape fitting the inner space 2710 of the barrel2730. In the practice of FNA, needle shaft is put inside the targettissue, the piston is pulled out from the inner space of the barrel anda vacuum is generated in the inner space of the barrel and transferredto the target tissue through the lumen of the needle shaft. The vacuumwill suck the target cells and tissue into the inner space 2711 of thechamber 2738.

FIG. 70B illustrates another embodiment of the tube 2722. In thisembodiment, the barrel 2730 is composed of upper 2701 and lower 2702portions. The two portions are mounted together by a joint 2703. Thejoint 2703 can be located at different location along the barrel; evencan be down across the tapered regions 2736 and/or non-tapered chamber2738. The inner surface of the barrel including the joint area should besmooth. The methods used to form the joint including 1) directconnection of the two portions by gel, glue, ultrasound, UV light andother methods; After finishing the sample collection, the two portionscan be separated from each other by a pushing force targeted on thejoint area or other methods; 2) different methods to form a joint basedon the principles described in FIGS. 57, 58, 59 and 60.

FIG. 70C illustrates a embodiment of the modified tube 2722. A supporttube 2719 is also added into the device.

FIG. 70D illustrates a different embodiment of tube 2722. In thisembodiment, the plug 2725 A is employed to replace the flat floor 2725.Different methods of connecting the plug 2725A to the chamber 2738,based on the principles described in FIGS. 57, 58, 59 and 60, may beemployed.

The tube 2722 has three specific functions. First, the tube 2722 worksas a fine needle aspiration device. In the practice of FNA, needle shaft2704 is put inside the target tissue, the piston 2705 is pulled out fromthe inner space 2710 of the barrel and a vacuum is generated in theinner space of the barrel and transferred to the target tissue throughthe lumen of the needle shaft. The vacuum will suck the target cells andtissue into the inner space 2711 of the chamber 2738.

Second, the tube 2722 works as a specimen collector to store thecollected material. The collected materials run through the shaft 2704and directly get into the inner space 2710 of the tube. This processeliminates any possible loss of the collected material during theirjourney from a shaft to the hub of conventional needle and to theconnecting joint between the hub and a collector and a possible nippleof the collector and finally reaches the collecting space of a collectorsuch as the barrel of a syringe.

Third, the tube 2722 works as a device where the collected materials maybe directly used to make a cell block. Collected materials from a FNAprocedure using this device will be directly stored in the inner space2710 and ideally in the inner space 2711 of the chamber 2738. Afterfinishing FNA procedure, the needle shaft will be amputated by aspecific tool similar to a vice, pincer or pliers. In this amputationprocess, the distal end of the shaft will be de-sharpened and the lumenof the shaft will be sealed either by a direct force which pushes thewall of the shaft close to each other and finally seal the lumen, or byfilling the lumen with special materials such as wax, gel, tape, orrubber or others. The supporting tube 2719 will be used to cover thebottom portion of the tube 2722. If the collected materials is less than300 micro liter (ul), a adequate amount of matrix will be added into theinner space 2711 to make a cell-matrix mixture G, the tube 2722 will beput at a lower temperature such as on ice or in a cooler for a fewminutes and the mixture G will be relatively hardened. At this moment,the G will be moved out from the inner space 2711 of the chamber and betransferred to a specific cassette as described in FIGS. 10, 11 and 67by a specific transfer tube as described in FIGS. 10, 11, 19 (A, B, C)and 68 or forceps 80 as described in FIG. 66. If the collected specimenhas a large volume (>400 ul), the tube will be put into a specialcentrifuge to spin down the cells to the flat floor of the chamber 2738,the supernatant will be moved out by different methods including apipette or a portable version of a vacuum device 14 as described inFIGS. 4 and 5. The remaining cell pellet will be used to make a cellmatrix mixture G using the method as described above.

In another embodiment as described in FIG. 70B, after collecting thespecimen, the lower portion 2702 of the barrel 2730 will be separatedfrom the upper portion 2701 from the joint 2703, and the lower portionwill be used for the cell block preparation as described above.

In another embodiment as described in FIG. 70D, after making a mixtureG, instead of transferring G from the chamber by a transfer tube orforceps, the plug 2725 A can separated from the chamber 2738, and themixture G will be put into a chamber 54A of a specifically designedcassette 30 A as described in FIG. 67 directly by pushing the G downthrough the lower opening of the chamber using a tamp.

This device can also be used in bone marrow aspiration. In current bonemarrow aspiration procedure, a specific bone marrow biopsy device (suchas LEE-LOK manufactured for LEE Medical, LTD.) with a long and wideneedle (11 gauge and 5½ inch) is used to penetrate the skin,subcutaneous soft tissue and bone. During this penetrating process, atamp is located inside the lumen of the needle to avoid a blockage ofthe lumen by the fragments of skin soft tissue or bone. Afterpenetrating through the bone and reaching into the bone marrow, the tampis moved out and the lumen of the needle will be opened, then a syringeis used to aspirate bone marrow from the lumen. And the collected bonemarrow will be used to make glass smears and cell block. There are twosteps to make a cell block of bone marrow in the current practice:aspiration by a syringe and transfer the collected bone marrow to adevice to make the cell block. In one embodiment of our tube 2722, theneedle shaft of the tube will be designed in an adequate size and lengthto fit and match with the lumen of the bone marrow biopsy needle. Andbone marrow can be directly aspirated into the inner space 2711 of thechamber of the tube 2722 and a cell block can be directly made asdescribed above.

The systems and methods described above enable a physician, for thefirst time, to perform a FNA and make a cell block using same device atthe same location in a very short time. This system has a few notableadvantages over the traditional FNA and cell blocking procedure: 1) itmaximally utilizes the collected material from a FNA procedure for adiagnostic purpose, 2) it takes much shorter time and much less steps tomake a cell block and shorten the turnaround time in practice, 3) ttsaves the time and labors of the technologists in making the cell blockusing traditional methods, 4) the size, shape and thickness of the cellblock can be controlled and the limited available materials can be usedefficiently to make enough sections for IHC or other special studies ifrequired.

FIG. 71 illustrates another embodiment of tube 2722. In this embodiment,the barrel 2701A of the tube 2722A does not have a tapered region and anon-tapered, reduced diameter chamber. The floor 2725A is either flat orslightly curved. A upper portion 2714A of A needle shaft 2704A is at thesame level of the floor 2725A and does not protrude into the inner spaceof the barrel. The bottom surface 2708A of the piston 2705 is in a shapewhich exactly matches the inner surface of the floor 2725A. In thiscase, when the piston is put inside the barrel, the bottom surface ofthe piston will completely attached on the inner surface of the floor2725A and there is no space present between the two surfaces.

The above tube embodiment 2722A has a several notable significances overa conventional syringe with an attached needle.

First, a metal needle shaft is directly extends to the bottom of a floorof a barrel with the inner lumen directly extends to the inner space ofthe barrel and there is of a needle and the inner space of the barrel.This setting may maximally reduce any possible liquid leakage andretaining material in these additional connections and joints. Whenusing this device to deliver a liquid medicine or chemical, it will bemore accurate to deliver a right dosage than a conventional syringe.Such an accurately delivery may be critical in some clinical scenario,for example, if the delivered medicine has a strong effects and sideeffects, or it is toxic, or it is very rare and very expensive.

Secondly, manufacture of this device may be much simpler and cheaper.Thirdly, it may be easier and friendlier to use since it is ready to beused, instead of opening a syringe and a needle separately and thenassemble them together to use.

Fourthly, as a disposable device, it can be designed to have twofunctions: 1) it is filled with a specific volume of a specific liquidmedicine or chemical and the distal opening of the shaft is coveredusing a specific method and it is ready to be used, in this way, itworks as a specific container of the specific medicine or chemical; 2)it works as a syringe to directly deliver the pre-filled medicine or thechemical to its target, instead of assembling a syringe with a needleand suck the medicine into the syringe first before deliver the medicineinto the target.

Although the present disclosure has been described with reference toexample embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the disclosed subject matter. For example, althoughdifferent example embodiments may have been described as including oneor more features providing one or more benefits, it is contemplated thatthe described features may be interchanged with one another oralternatively be combined with one another in the described exampleembodiments or in other alternative embodiments. Because the technologyof the present disclosure is relatively complex, not all changes in thetechnology are foreseeable. The present disclosure described withreference to the example embodiments.

1. A method of preparing a cytoblock comprising: a. preparing a cell pellet in a centrifuge tube having a relatively flattened floor; b. combining the prepared cell pellet with a matrix material to form a cell mixture; c. processing the cell mixture in a tissue cassette having at least one integrally formed chamber; and d. embedding the processed cell mixture in an embedding block.
 2. The method of claim 1, wherein the preparing further comprises: a. depositing cell material in the centrifuge tube; b. adding a predetermined amount of fixative to the centrifuge tube; c. placing the centrifuge tube in a centrifuge device to create the cell pellet and supernatant; and d. removing the supernatant from the centrifuge tube.
 3. The method of claim 1, wherein the centrifuge tube further comprises a tapered region and a non-tapered, reduced diameter bottom region.
 4. The method of claim 3, wherein the at least one integrally formed chamber is compatible in size and shape with the bottom region of the centrifuge tube.
 5. The method of claim 1, wherein the tissue cassette includes a removable insert having at least one integrally formed chamber.
 6. The method of claim 3, wherein the integrally formed chamber is compatible in size and shape with the bottom region of the centrifuge tube.
 7. The method of claim 1, wherein the preparing further comprises depositing cell material in a centrifuge tube containing a predetermined amount of fixative, placing the centrifuge tube in a centrifuge device to create the cell pellet and supernatant; and removing the supernatant from said centrifuge tube.
 8. The method of claim 7, wherein the cell material is obtained by fine needle aspiration.
 9. The method of claim 7, wherein the cell material is obtained by endoscopy.
 10. The method of claim 7, wherein the cell material is obtained by lavage.
 11. The method of claim 7, wherein the cell material is obtained by a catheter.
 12. The method of claim 1, wherein the combining further comprises: a. providing a matrix container containing the matrix material; b. heating the matrix container to liquefy the matrix material; c. transferring the cell pellet from the centrifuge tube to the matrix container; d. mixing the cell pellet and the matrix material to create a suspension; and e. cooling the matrix container to solidify the matrix material and create the cell mixture.
 13. The method of claim 1 wherein the combining further comprises: a. providing a matrix container containing the matrix material, the matrix container having the form of a syringe; b. heating the matrix container to liquefy the matrix material; c. depositing a predetermined amount of liquefied matrix material into the centrifuge tube; d. mixing the cell pellet and the matrix material to create a suspension; and e. cooling the centrifuge tube to solidify the matrix material and create a cell mixture.
 14. The method of claim 13, wherein the processing further comprises transferring the cell mixture from the matrix container to a tissue cassette and processing the tissue cassette.
 15. The method of claim 13, wherein the embedding further comprises: a. transferring at least a portion of the cell mixture from the tissue cassette to an embedding block made of wax and formed with at least one hole therein to receive the at least a portion of the cell mixture; b. placing an embedding tray over the embedding block and inverting the embedding tray and embedding block; c. warming the embedding tray and embedding block to at least partially melt the embedding block and embed the cell mixture in the embedding block; and d. cooling the embedding tray and embedding block to solidify the embedding block.
 16. The method of claim 13, wherein the embedding further comprises: a. transferring at least a portion of the cell mixture from the tissue cassette to an embedding tray, the embedding tray being provided with a partitioned insert formed with at least one partition therein to receive the at least a portion of the cell mixture; b. depositing a predetermined amount of embedding material into the embedding tray to embed the cell mixture into an embedding block, said embedding material being heated to liquefy the embedding material; and c. cooling the embedding tray to solidify the embedding block.
 17. The method of claim 1, wherein the embedding further comprises: a. transferring at least a portion of the cell mixture from the tissue cassette to an embedding tray, the embedding tray provided with liquefied embedding material therein and a portioned insert formed with at least one partition therein to receive the at least a portion of the cell mixture; and b. cooling the embedding tray to create a solidified embedded block.
 18. The method of claim 1, wherein the tissue cassette includes a fixed insert having at least one integrally formed chamber. 